TGF-betas in breast cancer progression

在2009财年，我们继续主要关注于阐明tgf - β活性从肿瘤抑制因子到促进展因子转换的肿瘤细胞自主成分。在这个项目中有两个主要的努力领域。在第一篇文章中，我们提出了tgf - β是癌症干细胞动力学的关键调节因子的假设，并且这种活性是其在癌变早期作为肿瘤抑制因子的核心作用。第二，我们正在使用整合的基因组方法来鉴定在癌症进展过程中导致tgf - β反应转换的分子决定因素，特别关注信号转导成分Smad3作为这一过程的关键介质。对于本研究的两个部分，我们的主要发现平台是基于MCF10A人乳腺上皮细胞系的乳腺癌进展的异种移植模型。我们之前已经证明，在该模型中，tgf - β从肿瘤抑制因子转换为促进展因子，并且进展系列的不同细胞系之间的高度遗传相关性为我们提供了一个异常高的信号-噪声系统，在该系统中可以解决tgf - β转换的潜在机制。然而，该模型的关键发现在可能的情况下在其他小鼠模型和人类临床材料中得到验证。继我们之前发表的tgf - β可能调节癌症干细胞动力学的论证之后，我们在过去的一年里专注于开发功能成像方法，这将使我们能够识别少数癌症干细胞（CSC）群体，从而分析其在微环境中与其他细胞和非细胞成分相互作用的背景下的行为。假设大多数CSC会表达一种或多种转录因子，这些转录因子可以在正常细胞中诱导或维持干细胞/多能性，我们已经生成了报道同时存在的干细胞相关转录因子Oct4和Sox2的构建体，我们已经证明这些报告体识别了富含CSC活性的细胞，包括在体内启动肿瘤发生的增强能力。利用表达不同荧光蛋白的tgf - β通路报告细胞，我们现在可以同时观察tgf - β通路在体外和体内的激活和干性。此外，我们正在开发和/或优化敏感的基于萤火虫的微蛋白质组学技术和单细胞基因表达分析工具，以解决这一少数群体的分子机制。有了这些工具，我们准备解决我们的假设，即tgf - β可能通过诱导或维持CSC静止，或通过影响自我更新和分化承诺之间的平衡来调节CSC动态。了解CSCs是如何被调节的对于开发更有效的癌症治疗方法至关重要，因为这些细胞对现有的治疗方法有很大的抵抗力，最终导致复发。在这个项目的第二部分，我们正在研究tgf - β反应从肿瘤抑制到肿瘤促进的分子机制。来自我们实验室和其他人的数据暗示了下游信号转导成分Smad3在这两种活动中。我们假设，在癌症进展过程中发生的遗传或表观遗传变化可能会改变smad3介导的tgf - β信号的读数，从而使促肿瘤活性占主导地位。为了解决这个问题，我们在MCF10模型中进行了全基因组染色质免疫沉淀，以确定Smad3靶基因对tgf - β治疗的反应。具体来说，我们专注于两个密切相关的细胞系；tgf - β在MCF10Ca1h中发挥肿瘤抑制作用，以及tgf - β在MCF10Ca1a中发挥促进展因子作用。启动子占用数据与全球基因表达数据的整合产生了与两种不同结果相关的基于smad3的核心基因签名。这些特征应该对开关过程背后的机制产生重要的见解，并将在基于基因表达的筛选中被利用，以发现可能逆转开关并恢复tgf - β肿瘤抑制活性的新化合物。

In FY09, we have continued to focus primarily on elucidating the tumor cell-autonomous components of the switch in activity of TGF-beta from tumor suppressor to pro-progression factor. There have been two main areas of effort on this project. In the first we are addressing the hypothesis that TGF-beta is a key regulator of cancer stem cell dynamics, and that this activity is central to its role as a tumor suppressor in the early stages of carcinogenesis. In the second, we are using integrated genomic approaches to identify molecular determinants that cause the TGF-beta response to switch during cancer progression, with a particular focus on the signal transduction component Smad3 as a key mediator of this process. For both parts of this study, we our main discovery platform is a xenograft model of breast cancer progression based on the MCF10A human breast epithelial cell line. We have previously demonstrated that TGF-beta switches from tumor suppressor to pro-progression factor in this model, and the high degree of genetic relatedness between the different cell lines of the progression series gives us an exceptionally high signal-to-noise system in which to address mechanisms underlying the TGF-beta switch. However, key findings from the model are validated in additional mouse models and human clinical material where possible. Following up on our previously published demonstration that TGF-beta may regulate cancer stem cell dynamics, we have focused over the past year on developing functional imaging approaches that will allow us to identify the minority cancer stem cell (CSC) population, and thus analyze its behavior in the context of interactions with other cellular and acellular components of the microenvironment. Starting with the assumption that most CSCs will express one or more of the constellation of transcription factors that can induce or maintain stemness/ pluripotency in normal cells, we have generated constructs that report on the simultaneous presence of the stem-related transcription factors Oct4 and Sox2, and we have shown that these reporters identify cells that are enriched for CSC activities, including an enhanced ability to initiate tumorigenesis in vivo. Using a TGF-beta pathway reporter expressing a different fluorescent protein, we can now simultaneously visualize TGF-beta pathway activation and stemness in vitro and in vivo. Furthermore, we are developing and/or optimizing sensitive Firefly-based microproteomic techniques and single cell gene expression analysis tools to address molecular mechanisms in this minority population. With these tools in hand, we are poised to address our hypothesis that TGF-beta may regulate CSC dynamics by inducing or maintaining CSC quiescence, or by influencing the balance between self-renewal and commitment to differentiation. Understanding how CSCs are regulated will be critical to development of more effective cancer therapies as these cells are largely resistant to existing therapeutic approaches, leading ultimately to relapse. In the second part of this project, we are addressing the molecular mechanisms that underlie the switch in TGF-beta response from tumor suppression to tumor promotion. Data from our lab and others implicates the downstream signal transduction component Smad3 in both these activities. We hypothesize that genetic or epigenetic changes that occur during cancer progression may alter the Smad3-mediated readout of the TGF-beta signal so that tumor promoting activities dominate. To address this question, we have performed genome-wide chromatin immunoprecipitation to identify Smad3 target genes in response to TGF-beta treatment in the MCF10 model. Specifically, we have focused on two closely related cell lines; MCF10Ca1h in which TGF-beta functions as tumor suppressor, and MCF10Ca1a in which TGF-beta functions as a pro-progression factor. Integration of the promoter occupancy data with global gene expression data has yielded core Smad3-based gene signatures that are associated with the two different outcomes. These signatures should yield important insights into mechanisms underlying the switch process, and will be exploited in gene expression based screens to find novel compounds that might reverse the switch and restore the tumor suppressor activities of TGF-beta.